Biologics are a category of medicinal products that are created by biologic processes for therapeutic purpose. Examples of biologics may include vaccines, blood or blood components, allergenics, somatic cells, gene therapies, tissues, therapeutic proteins, and living cells, among others. In most cases, the term “biologics” is used to specifically referred to a class of therapeutics that are produced by means of biological processes involving recombinant DNA technology. For instance, these biologics may include substances that are identical or nearly identical to the body's own key proteins, such as erythropoietin and insulin. Fusion proteins have also been used as biologics. For example, constructs have been used which contain cell surface receptors or other type of proteins linked to the immunoglobulin frame or an immunoglobulin fragment. Another example of biologics is monoclonal antibodies. These antibodies are typically “custom-designed” antibodies that are capable of binding to certain antigens in the body thereby blocking or modifying the unwanted biological function of the antigens.
In order to achieve a consistent efficacy in disease treatment, it is important to maintain a high level of homogeneity in the biologics. However, it is challenging to achieve a high level of homogeneity for biologics because biologic manufacturing is much more complex than traditional chemical synthesis. See e.g., Harris et al. 1993; Wan et al., 1999; Dorai H, et al. 2007; and Wen D, et al., 2009; A number of different technologies have been used to assess heterogeneity of biologics.
Mass spectrometry (MS or mass spec) based methods have been used in detection of sequence variants at the protein level. It has been reported that mass spectrometry (MS)-based peptide mapping was capable of detecting 1% to 27% of a Y376Q variant in a purified anti-Her2 antibody. See Harris et al. 1993. Through subcloning of the anti-Her2 antibody production Chinese hamster ovary (CHO) cell line followed by reverse transcription-polymerase chain reaction (RT-PCR), the authors were able to confirm that 10% of the subclones produced high levels of the Y376Q variant. Claverol and colleagues developed an electrophoresis-MSn method which could efficiently identify protein variants and post-translational modifications. See Claverol, et al., 2003. Using liquid chromatograph-MS/MS, Que et al. reported that 1% or lower of sequence variants in a monoclonal antibody could be detected. See Que et al., 2010.
Although MS-based methods have been reported to be capable of detecting single digit sequence variants, MS-based methods may miss certain type of variants such as unexpected, low-abundance, or variants with same/similar molecular weights. Typically, protein variants have to be isolated and enriched to a high level to achieve sensitive characterization. Moreover, MS-detected variants may not represent true genetic variations.
At the nucleic acid level, Sanger sequencing has traditionally played an important role in detecting sequence variants. In a typical process, the targeted region is amplified by PCR from a DNA template or by RT-PCR from an RNA template, followed by cloning, purification of plasmid clones and sequencing. For low-abundance variants, hundreds or thousands of clones need to be sequenced before a conclusion can be reached. PCR products can also be subject to sequencing directly. However, minor mutations (<30%) cannot be accurately detected. Overall, the process of PCR-cloning-sequencing is laborious and inaccurate in the detection of sequence variants, especially for low-abundance variants.